In general, the invention features compounds and methods for the treatment of bacterial infections, such as anthrax infection.
The etiologic agent of anthrax (Bacillus anthracis) is a potential threat as an agent of biowarfare or bioterrorism because exposure to aerosolized B. anthracis spores can be lethal to mammals, such as humans. The major virulence factors produced by this organism are the poly-D-glutamic acid capsule and anthrax toxin (ATx). Both the capsule and the toxin assist in colonization and immune evasion by the bacterium. ATx alone can cause death of the host. Vaccination against the toxin protects the host against infection.
Anthrax toxin is a member of the class of bacterial toxins termed A-B toxins. A-B toxins are composed of two moieties; the A moiety is the enzymic portion of the toxin that catalyzes the toxic effect upon a cytoplasmic target within a target cell. The B moiety binds to a cellular receptor and facilitates the translocation of the A moiety across the cell membrane into the cytoplasm of the cell.
The B moieties of A-B toxins from tetanus, botulinum, diphtheria and anthrax all form channels in membranes. It has been hypothesized that these channels might act as the conduit for the membrane translocation of the A moiety. The A and B moieities of anthrax toxin are secreted from the bacterial cell as distinct polypeptides. The A and B subunits of other A-B toxins are produced as single chain polypeptides or as separate chains that are assembled into oligomeric toxins before release from the bacteria. There are two alternative A subunits of anthrax toxin called edema factor (EF) and lethal factor (LF). Noncovalent complexes of EF or LF and the B subunit, protective antigen (PA), are called edema toxin and lethal toxin, respectively. PA facilitates the translocation of both EF and LF across membranes.
PA is secreted as an 83 kDa monomeric polypeptide. Monomeric PA binds to a mammalian cell surface receptor and is proteolytically cleaved. The C-terminal 63 kDa fragment (PA63) remains bound to the cell and the N-terminal 20 kDa (PA20) dissociates from PA63. This proteolytic cleavage and subsequent dissociation of PA20 confer two new properties on PA63: (1) the ability to oligomerize into a ring-shaped heptameric SDS-dissociable structure termed prepore and (2) the ability to bind EF and LF. Oligomers containing PA63-EF, PA63-LF, or a combination of PA63-EF and PA63-LF are endocytosed and trafficked to an acidic compartment, where the PA63 prepore inserts into the membrane and forms a pore. During or after pore formation, EF and LF are translocated across the endosomal membrane into the cytoplasm. EF is a calmodulin-dependent adenylate cyclase which may protect the bacteria from destruction by phagocytes. LF is a metalloprotease that can kill macrophages or, at lower concentrations, induce macrophages to overproduce cytokines, possibly resulting in death of the host.
A crucial step in this intoxication pathway is pore formation by PA. Low pH serves as the trigger for conversion of the PA63 prepore to the pore. This conversion is accompanied by a transformation of the oligomer from an SDS-dissociable to an SDS-resistant state and formation of a transmembrane 14-strand β-barrel. These steps are believed to be necessary for translocation of EF and LF across the endosomal membrane and, thus, toxin action.